Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi
ilustraciones, fotografías, graficas
- Autores:
-
Chacón Gómez, Miguel Esteban
- Tipo de recurso:
- Fecha de publicación:
- 2021
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/81733
- Palabra clave:
- 570 - Biología::572 - Bioquímica
Parásitos
Parasites
Proteínas recombinantes
Anticuerpos
Bioinformática
Membrana
Intracelular
Ensayos de complementación
Recombinant protein
Bioinformatics
Antibodies
Membrane
Intracellular
Complementation assays
Tripanosomiasis
Biotechnology
Biotecnología
- Rights
- openAccess
- License
- Atribución-NoComercial-CompartirIgual 4.0 Internacional
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Universidad Nacional de Colombia |
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| dc.title.none.fl_str_mv |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| dc.title.translated.spa.fl_str_mv |
Evaluation of a NAD+ carrier candidate in the protozoan parasite Trypanosoma cruzi |
| title |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| spellingShingle |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi 570 - Biología::572 - Bioquímica Parásitos Parasites Proteínas recombinantes Anticuerpos Bioinformática Membrana Intracelular Ensayos de complementación Recombinant protein Bioinformatics Antibodies Membrane Intracellular Complementation assays Tripanosomiasis Biotechnology Biotecnología |
| title_short |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| title_full |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| title_fullStr |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| title_full_unstemmed |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| title_sort |
Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi |
| dc.creator.fl_str_mv |
Chacón Gómez, Miguel Esteban |
| dc.contributor.advisor.none.fl_str_mv |
Ramírez Hernández, Maria Helena |
| dc.contributor.author.none.fl_str_mv |
Chacón Gómez, Miguel Esteban |
| dc.contributor.researchgroup.spa.fl_str_mv |
Libbiq Un |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica |
| topic |
570 - Biología::572 - Bioquímica Parásitos Parasites Proteínas recombinantes Anticuerpos Bioinformática Membrana Intracelular Ensayos de complementación Recombinant protein Bioinformatics Antibodies Membrane Intracellular Complementation assays Tripanosomiasis Biotechnology Biotecnología |
| dc.subject.other.spa.fl_str_mv |
Parásitos |
| dc.subject.other.eng.fl_str_mv |
Parasites |
| dc.subject.proposal.spa.fl_str_mv |
Proteínas recombinantes Anticuerpos Bioinformática Membrana Intracelular Ensayos de complementación |
| dc.subject.proposal.eng.fl_str_mv |
Recombinant protein Bioinformatics Antibodies Membrane Intracellular Complementation assays |
| dc.subject.unesco.eng.fl_str_mv |
Tripanosomiasis Biotechnology |
| dc.subject.unesco.spa.fl_str_mv |
Biotecnología |
| description |
ilustraciones, fotografías, graficas |
| publishDate |
2021 |
| dc.date.issued.none.fl_str_mv |
2021 |
| dc.date.accessioned.none.fl_str_mv |
2022-07-25T12:33:44Z |
| dc.date.available.none.fl_str_mv |
2022-07-25T12:33:44Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/TM |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/81733 |
| dc.identifier.instname.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.identifier.reponame.spa.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.unal.edu.co/ |
| url |
https://repositorio.unal.edu.co/handle/unal/81733 https://repositorio.unal.edu.co/ |
| identifier_str_mv |
Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
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spa |
| language |
spa |
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RedCol LaReferencia |
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Atribución-NoComercial-CompartirIgual 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Ramírez Hernández, Maria Helenae4ff3d503c25c95f30fecf7215f5065eChacón Gómez, Miguel Estebandd51e755c143dd105b11a20ad58788bfLibbiq Un2022-07-25T12:33:44Z2022-07-25T12:33:44Z2021https://repositorio.unal.edu.co/handle/unal/81733Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, fotografías, graficasTrypanosoma cruzi causa la enfermedad de Chagas, patología distribuida globalmente y carente de tratamientos efectivos, lo que hace pertinente la búsqueda de estrategias alternas de control. La dinámica del dinucleótido de nicotinamida y adenina (NAD+) es determinante en la homeostasis celular, y, las proteínas que participan en ella son promisorios blancos farmacológicos. La síntesis del NAD+ es citosólica en T. cruzi, por lo que un sistema intracelular de distribución debe existir; proteínas de la Familia de Transportadores Mitocondriales (MCF) cumplen esta función en eucariotas, y se espera que el parásito cuente con homólogos capaces de movilizar al dinucleótido. Tres secuencias candidato a transportador de NAD+ de T. cruzi fueron caracterizadas; el estudio in silico de las proteínas TcNdt1 y TcNdt2 mostró que estructuralmente conservan características de la MCF, y por docking molecular se determinó que están dotadas de elementos capaces de interactuar específicamente con el dinucleótido. Por su parte, el candidato TcNdt3 es una proteína atípica de la MCF con una duplicación de sus elementos estructurales. En los 3 candidatos se predice una localización mitocondrial o glicosomal, al igual que la presencia sitios blanco de modificaciones postraduccionales. Ensayos de complementación realizados con las cepas Δndt1 y Δndt2 de Saccharomyces cerevisiae mostraron que TcNdt1 y TcNdt2 reestablecen el crecimiento rezagado de los mutantes en medio no fermentable, comprobando su actividad transportadora de NAD+. De forma complementaria, se desarrolló el sistema de expresión MISTIC que media la inserción de proteínas en membranas de E. coli, y se adelantaron ensayos piloto de transporte del dinucleótido. Empleando un antígeno recombinante generado en E. coli se produjeron IgY que permitieron la detección por inmunofluorescencia de la TcNdt2 endógena sobre epimastigotes de T. cruzi, indicando que presenta localización posiblemente glicosomal asociada a tráfico vesicular. Adicionalmente, se evaluó la expresión de un fragmento de la RVG del virus de la rabia en S. cerevisiae, donde fue posible obtener un patrón de reconocimiento diferencial en la inmunodetección, el cual es atribuible a la expresión de la recombinante en el modelo eucariota. Los resultados obtenidos en este estudio constituyen un aporte importante para entender la simplificación en el metabolismo del NAD+ en parásitos intracelulares, y sus relaciones con el hospedero. (Texto tomado de la fuente)Trypanosoma cruzi is the etiological agent of Chagas disease, a disease that has a worldwide distribution and lacks effective treatments, making it pertinent to look for alternate control strategies. The nicotinamide-adenine nucleotide (NAD+) dynamics determines cell homeostasis, and the proteins involved in it are interesting pharmacological targets. NAD+ synthesis is cytosolic in T. cruzi, and therefore, an intracellular distribution system should exist; proteins belonging to the Mitochondrial Carrier Family (MCF) fulfill this role in eukaryotes, ant it is expected that the parasite has MCF homologues able to transport the dinucleotide. Three NAD+ carrier candidate sequences from T. cruzi were studied; in silico analysis of the TcNdt1 and TcNdt2 protein showed that typical structural features from the MCF are conserved in these proteins, and, through molecular docking, it was found that they are both endowed with structural elements able to interact specifically with the dinucleotide. On the other hand, the TcNdt3 candidate is an atypical MCF protein that shows a complete duplication of its structural elements. The 3 candidate sequences are predicted to have a mitochondrial or glycosomal localization, and throughout the TcNdt1, TcNdt2 and TcNdt3 sequences post-translational modification sites are predicted. Complementation assays carried out with the Saccharomyces cerevisiae mutant strains Δndt1 and Δndt2, showed that the TcNdt1 and TcNdt2 sequences reestablish yeast cell growth on a non-fermentable media, supporting that the T. cruzi proteins are functional NAD+ carriers. As a complementary approach, the MISTIC expression system, that mediates recombinant protein insertion in E. coli membranes, was developed, and pilot dinucleotide transport assays were performed. Using a recombinant antigen produced in E. coli, specific IgY against TcNdt2 were raised, which allowed for the endogenous carrier recognition through immunofluorescence on T. cruzi epimastigotes, showing that the NAD+ carrier has a glycosomal localization, linked to the vesicular transport. Further, the expression of a recombinant fragment derived from the rabies virus RVG protein was evaluated in S. cerevisiae, and at the immunodetection a differential recognition pattern was obtained, which can be attributed to the recombinant protein production in the eukaryotic system. The results obtained in this study make for an important contribution for the understanding of the NAD+ metabolism simplification that occurred in the intracellular parasites, and for the understanding of the host-parasite interactions.MaestríaMagíster en Ciencias - BioquímicaBioquímica y Biología Molecular de Parásitosxix, 112 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaDepartamento de QuímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - BioquímicaParásitosParasitesProteínas recombinantesAnticuerposBioinformáticaMembranaIntracelularEnsayos de complementaciónRecombinant proteinBioinformaticsAntibodiesMembraneIntracellularComplementation assaysTripanosomiasisBiotechnologyBiotecnologíaEvaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruziEvaluation of a NAD+ carrier candidate in the protozoan parasite Trypanosoma cruziTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMRedColLaReferencia(1) Coura, J. 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In Methods in Enzymology; Elsevier, 2014; Vol. 536, pp 133–147. https://doi.org/10.1016/B978-0-12-420070-8.00012-X.EstudiantesInvestigadoresMaestrosORIGINAL1026581504.2021.pdf1026581504.2021.pdfTesis de Maestría en Ciencias – Bioquímicaapplication/pdf6412116https://repositorio.unal.edu.co/bitstream/unal/81733/3/1026581504.2021.pdfc69ae479ea06cbabc52e43daa036c247MD53LICENSElicense.txtlicense.txttext/plain; charset=utf-84074https://repositorio.unal.edu.co/bitstream/unal/81733/4/license.txt8153f7789df02f0a4c9e079953658ab2MD54THUMBNAIL1026581504.2021.pdf.jpg1026581504.2021.pdf.jpgGenerated Thumbnailimage/jpeg5092https://repositorio.unal.edu.co/bitstream/unal/81733/5/1026581504.2021.pdf.jpg9f9e1355427d41263a123aa14e36b240MD55unal/81733oai:repositorio.unal.edu.co:unal/817332024-08-06 23:10:06.628Repositorio Institucional Universidad Nacional de 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